Tissue factor (TF).sup.1 is a 45-kDa integral membrane glycoprotein that is an essential cofactor in initiating the extrinsic pathway of blood coagulation. In response to blood vessel injury, TF, which is produced constitutively by cells that are separated from blood by the vascular endothelium, gains access to the plasma. Plasma factor VII or VIIa binds TF and the resulting factor VIIa-TF complex activates factors X to Xa and IX to IXa. This eventually leads to the generation of thrombin and the formation of a fibrin clot. TF-induced blood coagulation is primarily regulated by tissue factor pathway inhibitor (TFPI), a 42-kDa plasma glycoprotein also referred to as lipoprotein-associated coagulation inhibitor (LACI) and extrinsic pathway inhibitor (EPI). TFPI contains an acidic amino-terminal domain followed by three tandem Kunitz-type protease inhibitory domains and a basic carboxy-terminal domain. Inhibition of TF-induced blood coagulation by TFPI involves a two step reaction leading to the formation of a quaternary factor Xa-TFPI-factor VIIa-TF complex. In the first step factor Xa binds to the second Kunitz domain of TFPI and in the second step, TFPI-factor Xa binds to the TF-factor VIIa complex through an interaction between the first Kunitz domain of TFPI and factor VIIa (reviewed in Refs. 1,2).
A wide range of plasma TFPI concentrations is found in normal individuals with a mean of .about.2.5 nM (3). Greater than 90% of this TFPI is bound to lipoproteins (low density lipoprotein&gt;high density lipoprotein&gt;very low density lipoprotein), (4,5). Plasma TFPI levels increase several fold following the infusion of heparin (6,7). TFPI is thought to be released from the vascular endothelium where it may be bound to heparan sulfate or glycosaminoglycans.
Several animal studies have shown that recombinant TFPI is effective against TF-induced coagulopathy (8), prevents arterial thrombosis (9), and reduces mortality from bacterial septic shock (10). Pharmacokinetic studies (11) following an intravenous bolus injection of recombinant TFPI in rabbits have shown that TFPI clearance from the plasma is a biphasic process with half-lives of 2.3 min and 79 min. The primary organs involved in TFPI clearance are the liver and kidney (especially the outer cortex).
The low density lipoprotein receptor-related protein/a.sub.2 -macroglobulin receptor (LRP) and glycoprotein 330 (gp330) are two members of the low density lipoprotein receptor family involved in the endocytosis of several circulating plasma proteins. The endocytic function of LRP appears to be predominantly in the liver whereas that of gp330 is in the kidney (reviewed in Ref. 12). LRP and gp330 bind similar ligands including complexes between plasminogen activator inhibitor type 1(PAI-1) and tissue-type (t-PA) or urokinase-type (u-PA) plasminogen activators, .beta.-migrating very low density lipoproteins (.beta.VLDL) complexed with apolipoprotein (apo) E, lipoprotein lipase, and lactoferrin (13-15). In addition, LRP binds .alpha..sub.2 -macroglobulin-protease complexes .alpha..sub.2 m*) (16-18) and Pseudomonas exotoxin A (19). A 39-kDa protein, also termed receptor-associated protein (RAP), copurifies with both LRP and gp330 (16,20). This 39-kDa protein is a potent inhibitor of all known ligand interactions with LRP and gp330, as shown by ligand blotting experiments and by binding and uptake experiments in cultured cells (13,14,19,21-24). It has been recently reported that intravenous administration of the 39-kDa protein to rats prolonged the plasma half-life of t-PA from 1 min to .about.6.about.9 min. It was also found that the 39-kDa protein itself was rapidly cleared from the circulation, with the liver and outer cortex of the kidney being the primary sites of clearance (25). Although the in vivo physiological role of the 39-kDa protein at present is not clear, it has been postulated to function as a regulator of LRP and gp330 activity.